Nucleic acid bulges refer to regions of unpaired bases in a double-stranded nucleic acid molecule. These bulges have been known to take part in many important biological processes.
For example, ENA bulges form crucial motifs for specific nucleic acid-protein recognition and binding. It has been known that the human immunodeficiency virus transactivator protein Tat binds to a three-pyrimidine bulge in the response element TAR. See, e.g., Weeks et al., Science 249, 1281-1285 (1990). Nucleic acid bulges also produce frameshift mutations which can change the product of the protein translation and result in various disorders. According to one report, Myerowitz et al., J. Biol. Chem. 263, 18587-18589 (1988), approximately 70% of Ashkenazi Tay-Sachs disease is caused by a four-base pair insertion mutation in the HEX A gene encoding the xcex1-subunit of hexosaminidase A. Another disease, cystic fibrosis, is also caused by frameshift mutation. A three-base pair deletion (AF508) is commonly found among cystic fibrosis patients. Rommens et al., Am. J. Hum. Genet. 46, 395-396 (1990).
Comparative gel electrophoresis assay has been used to detect the presence of bulges in nucleic acids. This assay differentiates nucleic acids with and without bulges by their different mobility in gel. However, it can only provide information as to whether a nucleic acid contains a bulge. Thus, there exists a need for a detection method which can provide additional information, e.g., the location of a bulge in a nucleic acid.
One aspect of this invention relates to a metal complex of formula (I): 
Each of R1, R2, R3, R4, R5, R6, R7 and R8, independently, is hydrogen, alkyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, aminoalkyl, alkylcarbonylamino, alkylaminocarbonyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. Each of R2 and R3, and R6 and R7, independently, optionally join together to form a cyclic moiety which is fused with the two pyridyl rings to which R2 and R3, or R6 and R7 are bonded. The cyclic moiety, if present, is optionally substituted with alkyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, aminoalkyl, alkylcarbonylamino, alkylaminocarbonyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. Each of L1 and L2, independently, is xe2x80x94C(Ra) (Rb)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, or xe2x80x94N(Rc)xe2x80x94 and each of Ra, Rb, and Rc, independently, is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. M is a Co, Ni, Ru, Rh, Mn, Os, Ag, Cr, Zn, Cd, Hg, Re, Ir, Pt, or Pd ion, and the oxidation state of M is 0, 1, 2, 3, or 4. Each of X1 and X2, independently, is a labile ligand.
Examples of a metal complex of formula (I) include cobalt(II)(hexaazacyclophane)(trifluoroacetate)2, cobalt (II) (hexaazacyclophane) (H2O) (trifluoroacetate) ruthenium(II) (hexaazacyclophane) (trifluoroacetate)2, and manganese(II)(hexaazacyclophane)(trifluoroacetate)2.
Another aspect of this invention relates to a method of specifically cleaving a nucleic acid bulge. The method comprising contacting the bulge with a metal complex of formula (I), supra, where M is a Fe, Co, Ni, Ru, Rh, Mn, Os, Ag, Cr, Zn, Cd, Hg, Re, Ir, Pt, or Pd ion. In one embodiment, the method is performed in the presence of an oxidant, e.g., hydrogen peroxide, or in a medium having a pH values which ranges from 4-9.
In this disclosure, a nucleic acid bulge is a region in a double-stranded nucleic acid molecule (DNA or RNA), the region having at least one unpaired nucleotide and being flanked by two paired nucleotides. The nucleic acid bulge can contain 1-5 unpaired nucleotides (e.g., 1-3). Using nucleic acid substrate A in FIG. 1 as an example, the nucleic acid bulge present therein contains three unpaired nucleotides, i.e., T6, C7, and T8. This three-base bulge is flanked by two paired nucleotides, i.e., A5-T23 and G9-C22. In contrast, the C13-A18 hairpin loop, which is also present in substrate A, is not a bulge as the unpaired nucleotides are only connected to one paired nucleotide, i.e., C12-G19. A nucleic acid bulge can also contain two nucleotides. See the bulge present in substrate D which is formed of two unpaired nucleotides, i.e., C6 and T7.
A salt of a metal complex of formula (I) is also within the scope of this invention. Note that a metal complex of formula (I) can be positively charged. A salt of such a metal complex can be formed with an anionic counterion. Examples of counterions include fluoride, chloride, bromide, iodide, sulfate, sulfite, phosphate, acetate, oxalate, and succinate.
As described above, each of R2 and R3, and R6 and R7, independently, can join together to form a cyclic moiety. The cyclic moiety can contain 5 or 6 ring members and can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. For example, when the cyclic moiety formed by joining R2 and R3 is a benzene, it fuses with the two pyridine rings co which R2 and R3 are bonded, and the benzene ring and the two pyridine rings together form phenanthroline.
As used herein, alkyl is a straight or branched hydrocarbon chain containing 1 to 6 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and hexyl.
By xe2x80x9ccycloalkylxe2x80x9d is meant a cyclic alkyl group containing 3 to 8 carbon atoms. Some examples of cycloalkyl are cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl. Heterocycloalkyl is a cycloalkyl group containing 1-3 heteroatoms such as nitrogen, oxygen, or sulfur. Examples of heterocycloalkyl include piperidine, piperazine, tetrahydropyran, tetrahydrofuran, and morpholine.
In this disclosure, aryl is an aromatic group containing 6-12 ring atoms and can contain fused rings, which may be saturated, unsaturated, or aromatic. Examples of an aryl group include phenyl and naphthyl. Heteroaryl is aryl containing 1-3 heteroatoms such as nitrogen, oxygen, or sulfur. Examples of heteroaryl include pyridyl, furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, and benzothiazolyl.
Note that an amino group can be unsubstituted, mono-substituted, or di-substituted. It can be substituted with groups such as alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. Halo refers to fluoro, chloro, bromo, or iodo.
A labile ligand (i.e., X1 or X2) refers to a group which coordinates with less affinity to the metal ion (i.e., M) of a complex of formula (I) relative to the four pyridyl nitrogen atoms. Such ligand can therefore undergo rapid equilibrium with other labile ligands. Examples of a labile ligand include H2O, Cl, trifluoroacetate, or pyridine.
A metal complex of formula (I) possesses unexpectedly high specificity toward nucleic acid containing a bulge structure. As described above, a nucleic acid with such a structure is associated with various disorders. A metal complex of formula (I) can therefore be used in a diagnostic kit for detecting nucleic acid bulge-associated disorders.
Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.